Prolactin is a hormone which dramatically affects fertility and lactation and which therefore plays a crucial role in both the creation and nurturing of offspring. In addition, prolactin also plays a more subtle role in the functioning of many tissues such as the immune system, the liver and the endocrine pancreas, which need to adjust to the demands of pregnancy and lactation. It is therefore of immense importance for us to understand how total levels of prolactin are regulated at the different stages of reproduction and further, because there is not known to be a family of prolactin molecules, how the composition of secreted prolactin may change. The primary influences on prolactin secretion are hypothalamic/posterior pituitary, autocrine/paracrine, and gonadal. In this project we propose 1) further study of autocrine inhibition of prolactin secretion in isolation, and 2) a study of how this integrates with controls from the hypothalamus and gonads. For the latter we will determine the affects of a major hypothalamic factor, dopamine, and a major gonadal hormone, estrogen, ont he autocrine feedback system. In this regard, we hypothesize that dopamine inhibition of secretion is independent of, whereas estrogen stimulation of secretion works through, adjustments in the autocrine mechanism. Also, because autocrine adjustments are accomplished via shifts in the phosphorylation state of the forms secreted, we will in the process determine whether dopamine and estrogen affect the composition of secreted prolactin. When studying the autocrine system in isolation, dose-response curves to preparations of non-phosphorylated and phosphorylated prolactin and physiological combinations thereof will allow us to determine what settings (proportion of non-phosphorylated to phosphorylated prolactin secreted) are consistent with significant autocrine control so that we may predict, based on our in vivo analyses, when this mechanism is most important and when other controlling factors must predominate. For these dose response studies we will use GH3 cells and primary rat pituitary cells in culture. After analysis of the natural phosphorylation sites, phosphorylated prolactin for these studies will be produced in vitro from recombinant rat prolactin. The effects of dopamine and estrogen will be assessed in vitro on primary cultures and the effects of a dopamine agonist, bromocryptine, and an antagonist, haloperidol, will be assessed using a combined in vivo in vitro approach.